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Biomedical subjects

E Diener

Publications and source records attributed to E Diener.

At least 109 records · Page 6Linked to original sources
PubMed1971

The separation of different cell classes from lymphoid organs. IV. The separation of lymphocytes from phagocytes on glass bead columns, and its effect on subpopulations of lymphocytes and antibody-forming cells.

Four separate effects can be demonstrated when lymphoid cell suspensions are passed through columns of siliconed glass beads. (a) A temperature-dependent "active adherence" of phagocytic cells, such as macrophages and polymorphs. (b) A temperature-independent and selective trapping by "physical adherence" of particular classes of lymphoid cells, including certain antibody-forming cells. (c) A "size-filtration" effect that traps larger cells, but only becomes significant with beads below 100 micro in diameter. (d) A selective retention of damaged cells, which occurs with all columns under all conditions tested. An active adherence column technique has been developed to separate phagocytes from lymphocytes while minimizing selection within the lymphocyte population by physical adherence or size filtration. In less than 10 min at 37 degrees C it reproducibly produces a preparation of mouse spleen lymphocytes >500-fold depleted of active macrophages, and approximately 50-fold depleted of active polymorphs, with good over-all cell recoveries and cell viability. The lymphocyte fraction appears fully active in its ability to initiate immune responses to at least two different antigens, but is changed in over-all composition and selectively depleted in certain classes of antibody-forming cells.

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PubMed1970

Antibody-mediated suppression of the immune response in vitro. II. A new approach to the phenomenon of immunological tolerance.

Immunological tolerance to H antigens of Salmonella adelaide may be induced in vitro by the exposure of mouse spleen cells for 6 hr to an immunogenic dose of polymerized flagellin in the presence of low concentrations of specific antibody. Such antibody-mediated tolerance requires an optimal antigen: antibody ratio for its induction. A shift in this ratio in favor of the antibody concentration results in failure of tolerance induction and leads to immune suppression commonly known as antibody-mediated feedback inhibition which is not analogous to immunological tolerance. Fragment A of flagellin fails to induce immunological tolerance in vitro. Tolerance to polymerized flagellin may however be induced in vitro, provided the spleen cells are exposed to fragment A in the presence of specific antibody for 6 hr. The results are discussed in the light of current theories of the mechanism of tolerance induction.

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PubMed1970

The role of nonlymphoid accessory cells in the immune response to different antigens.

Tissue culture techniques were combined with cell separation procedures to investigate the cellular requirements for a response to antigen, leading to the production of antibody-forming cells. Mouse spleen was dissociated, and the cells were separated into various groups on the basis of density, size, and active adherence. The ability of fractions to initiate a response in vivo, on transfer to an irradiated recipient, was compared to the response in vitro; and this ability was correlated with the presence or absence of phagocytic cells. Two different antigens were studied, sheep erythrocytes (SRC) and polymerized bacterial flagellin (POL). Density distribution analysis of spleen showed a wide density range of cells responding to both antigens in vivo. The same fractions responded to POL in vitro as in vivo. By contrast, only the light density regions responded in vitro to SRC. Response occurred in regions of overlap between lymphocytes and phagocytic macrophages. Separation by active adherence on columns of large glass beads gave a preparation containing large, medium, and small lymphocytes but no detectable phagocytic macrophages and very low levels of phagocytic polymorphs. This lymphocyte preparation responded to both antigens in vivo. In vitro it gave a full response to POL, but no response to SRC. Addition of a small quantity of the adherent fraction, enriched for phagocytic cells, restored response to SRC. The use of strain-specific antisera in a mixed culture containing a C57 phagocytic fraction and CBA lymphocytes showed that the lymphocyte fraction contributed the precursors of the final antibody-forming cells. The accessory cells from C57 spleen banded in the light regions of the density gradient where phagocytic macrophages were found. Irradiated spleen cells also activated the lymphocyte preparation, suggesting that the irradiated host provided the accessory cells for the in vivo response to SRC. Small lymphocytes were purified from spleen by the small glass bead size filtration technique. This sample of small lymphocytes responded less well to POL than the total lymphocyte population, but it responded as well in vitro as in vivo. The small lymphocyte preparation responded in vivo to SRC but not in vitro. Addition of a small quantity of the phagocyte-rich fraction from adherence columns restored the in vitro response to SRC. The results indicated that phagocytic cells are not required in the initiation of an immune response to POL. By contrast some accessory cell, possibly a phagocytic macrophage, is required for a response to SRC. The basis for this marked difference is discussed.

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PubMed1970

Antibody-mediated suppression of the immune response in vitro. I. Evidence for a central effect.

Antibody-mediated suppression of the in vitro immune response to polymerized flagellin of Salmonella adelaide and to sheep erythrocytes was studied at the cellular level. Normal mouse spleen cells, preincubated in vitro with mixtures of antigen and antibody for short periods of time before being washed, did not respond to an optimal antigenic challenge in vitro, whereas similar cells treated with antibody alone gave a normal response. The degree of immune suppression was found to depend on the time of preincubation. Significant immune suppression could be induced in as short a time as 15 min, whereas profound suppression (90%) required the incubation of cells with mixtures of antigen and antibody for 4-6 hr. Mouse spleen cells treated similarly were also unable to respond subsequently to the antigen upon transfer to lethally irradiated hosts, as measured at both the level of the antigen-reactive cell and that of serum antibody production. These results were taken as evidence that in vitro an effect of antibody-mediated suppression occurred at the level of the immunocompetent cell. Similarities between immune tolerance and antibody-mediated suppression in vitro were described, and the significance of the findings discussed in the light of current concepts of the mechanism of antibody-mediated suppression.

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PubMed1969

Immunological tolerance in vitro: kinetic studies at the cellular level.

When normal mouse spleen, cells in suspension are cultured in vitro in the presence of polymer from S. adelaide flagellin, an immune response can be obtained as measured at the level of single antibody-forming cells. Cultures were stimulated with different doses of antigen, ranging from 0.2 ng to 3 microg/ml of tissue culture fluid and it was found that the peak number of approximately 500 antibody-forming cells per 10(6) harvested cells by day 4 was antigen dose dependent, 2-20 ng/ml being the optimal concentration. When more than 1 microg/ml of polymer from S. adelaide together with either 20 ng/ml of polymer from S. waycross or with 4 x 10(6) sheep erythrocytes were placed in the system, unresponsiveness to S. adelaide, but immunity to the other antigens occurred simultaneously. Cells made immunologically tolerant in vitro to S. adelaide H antigens were transferred into syngeneic lethally irradiated recipients and challenged with the same antigen. The adoptive immune capacity in these mice, as measured at the level of the immunologically competent cell was reduced by 80-90% as compared with relevant controls. Attempts to induce low zone tolerance in vitro were without success. To study the kinetics of tolerance induction in vitro, cells were cultured with tolerogenic doses of antigen for various periods of time, washed, and subsequently cultured with immunogenic doses of antigen for 4 days. It was found, that immunological tolerance may be induced to a significant degree in vitro within a period of 15 min. Similar results were obtained when spleen cells were exposed for various lengths of time to tolerogenic doses of antigen but at a temperature of 4 degrees C instead of 37 degrees C. The results are taken as suggestive evidence that the initial step in tolerance induction is related to the direct interaction between the surface of immune competent cells and antigen molecules.

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PubMed1969

A new method for the enumeration of antigen-reactive cells responsive to a purified protein antigen.

A new technique for the enumeration of antigen-reactive cells (ARC) responsive to the polymer antigen of S. adelaide flagellin (POL) is described for two strains of mice. Foci have been shown to be antibody dependent, may be mimicked by IgM as well as IgG antibodies, and contain specific antibody-forming cells (AFC). The use of POL offers a system unencumbered by relatively high numbers of background foci which, when present, appear to be basically different from those found using the SRBC antigen. The response of 1 antigen-reactive cell (ARC) focus/1 x 10(6) CBA(T6T6) mouse spleen cells is linearly related to the injected number between 1 x 10(6)-3 x 10(6) donor spleen cells and since 5% of injected cells remain in the spleen, there are an estimated 2400 ARC/spleen. The number of ARC foci does not increase significantly after the 5th postantigen day, and by the 8th day the AFC progeny of ARC have reached the maximum mean of 280 AFC/ARC focus. In response to increasing antigen concentrations, an initial rise in the number of AFC as well as ARC is observed, resulting in a relatively constant AFC/ARC ratio. This suggests that the number of ARC stimulated determines the total number of AFC produced under these conditions rather than a variable mitotic rate of the ARC offspring. The main significance of this technique is that it will allow a study of the kinetics of the ARC in the primary and secondary immune response as well as in immunological tolerance.

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PubMed1969

Antigen-reactive cells in normal, immunized, and tolerant mice.

The numbers of antigen-reactive cells (ARC) responding to a purified protein, the polymer of S. adelaide flagellin, have been assayed in cell populations derived from several lymphoid tissues of mice. The assay, which employs the cell transfer into lethally irradiated mice, indicates that there is a response of ARC in bone marrow in the absence of thymus cells. This suggests that the immune response to this protein antigen is not thymus dependent. The presence of relatively large numbers of ARC in Peyer's patches argues for their direct participation in the immune response in the adult mouse. The kinetics of ARC and antibody-forming cells in the early primary response employing the transfer system is described. The numbers of ARC declined during the first 2 days of the immune response, but by day 6 had increased to about five times the number in unprimed spleen cells. The rise is believed to be a result of the primary injection of antigen and therefore may be described as memory; however, these experiments have not been able to further elucidate any specific qualities of the "memory cell." Tolerance induction in C(57)BL/Brad mice produced by repeated injections of a cyanogen bromide digest of the antigen is described. The ARC or its precursor is shown to be the site of the lesion of tolerance by direct investigation.

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